TheEffectsofIsotre.noinonMG-63CancerCells

ColmParrishPi;sburghCentralCatholicHS

12thGrade

AnOverviewofCancerCells

•  Cancercellsarecellsthatgrowanddivideatanirregular,unregulatedpace

•  Apoptosisdoesnotoccurincancerouscells;muta.onsarepassedontothesubsequentgenera.on,eventuallyclusteringandformingtumors.

•  Tumorscanbemalignantorbenign

MG-63CancerCells

•  HumanOsteosarcomacells,anaggressiveformofbonecancer

•  Modelstheeffectsofvariableoncancercellprolifera.on

Isotretinoin•  A naturally occurring retinoic acid

•  Supplemented for treatment of severe acne

•  Can also be used in the treatment of skin cancer

•  Prescribed under the names Roaccutane(formerly Accutane), Amnesteem, Claravis and others

Controversy over Isotretinoin•  Very powerful teratogen

–  In 2005, the FDA responded with iPledge

•  A link between depression/anxiety has been suggested, but there is no causal evidence

PreviousStudies

•  Chemopreven.onofbasalcellcarcinomawithisotre.noin(Peck,Gross,Butkus,DiGiovanna)– Averagemaximumdoseof4.5mg/kg/day(Highconcentra.on),effec.veathighdose

•  Preven.onofskincancerinxerodermapigmentosumwiththeuseoforalisotre.noin(Kraemer,DiGiovanna,Moshell,Tarone,Peck)– Averagemaximumdoseof1.5mg/kg/day(Lowconcentra.on),resultsvariedbetweenpa.ents

Purpose

To determine the effects of Isotretinoin supplementation on

the proliferation of MG-63 cancer cells.

Hypotheses•  Null Hypothesis: Isotretinoin

supplementation will have no significant effect on the proliferation of MG-63 cancer cells

•  Alternative Hypothesis: Isotretinoin supplementation will significantly decrease the proliferation of MG-63 cancer cells.

Materials•  Isotretinoin solution

(Amnesteem)•  75mm2 tissue culture

treated flasks•  Twenty 25 mm2 tissue

culture treated flasks•  MG-63 Cancer Cell Line•  Trypsin-EDTA•  Pen/strep•  Power pipette+ sterile

macropipette tips (1 mL, 5 mL, 10, mL, 20 mL)

•  Micropipettes + sterile tips•  DMEM Serum - (4 mM L-

glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L sodium bicarbonate + [ 10% fetal bovine serum

for complete])•  75 mL culture flask•  Incubator•  Nikon Inverted Microscope•  Aspirating Vacuum Line•  Laminar Flow Hood•  Laminar Flow Hood UV

Sterilizing Lamp•  Labeling Tape•  Hemocytometer•  Sterile PBS•  Ethanol (70% and 100%)•  Distilled water

CulturingProcedure1.  A1mLaliquotofMG63cellsfromaCryotankwas

usedtoinoculate30mLof10%serumDMEMmediainone75mm2cultureflaskyieldingacelldensityofapproximately106to2x106cells.

2.  Themediawasreplacedwith15mLoffreshmediatoremovecryo-freezingfluidandincubated(37°C,5%CO2)for2daysun.lacelldensityofapproximately4x106to5x106cells/mLwasreached.

3.  Theculturewaspassedinto16T25flasksinprepara.onforexperimentandincubatedfor2daysat37°C,5%CO2.

CulturingProcedurecon’t4.  A`ertrypsiniza.on,cellsfromalloftheflaskswerepooled

into1common75mm2flask(celldensityofapproximately1millioncells/mL).

5.  0.1mLofthecellsuspensionwasaddedtoeight25mm2.ssueculturetreatedflaskscontaining5mLofDMEM(com)media,crea.ngacelldensityofapproximately105cellsperflask.

6.  TheIsotre.noinsolu.onwascreatedbydilu.ng40mgIsotre.noinin40mLof10%media

7.  Thecellswereincubatedat37°C,5%CO2fortheremainderofthestudy.

Coun.ngProcedure

8.  Days1and3– Usingoneflaskfromeachgroup,celldensi.esweredeterminedasfollows:•  Thecellsweretrypsinizedandcollectedintocellsuspension.

•  25µlaliquotsweretransferredtoaHemocytometerforquan.fica.on(eightcountsperflask).

TheEffectsofIsotre.noinonMG63Prolifera.on

0

50

100

150

200

250

Control Low(7.1x10^-4) High(1.59x10^-3)

Day1

Day3

Concentra.onofIsotre.noin

CellCo

untp

erflask(1000cells)

P-Value6.53E-12

P-Value0.00765

Dunne;’sTestResults

T-Crit T-Value Variation

Day 1 Low 2.67 14.679 Significant

Day 1 High 2.67 9.72 Significant

Day 3 Low 2.67 2.99 Significant

Day 3 High 2.67 3.11 Significant

Conclusions

Thealterna.vehypothesiswasaccepted,sugges.ngthatIsotre.noinsupplementa.onsignificantlydecreasestheprolifera.onofMG-63Cancercells

Limitations•  Hemocytometer counts can vary•  Cell Clumping•  Lag Time•  Low number of replicates and

concentrations

Limitations and Extensions•  Limited number of replicates and

concentrations•  Lag time •  Test the effects of Isotretinoin on

other cell lines (3T3, etc.)•  CyQUANTTM Cell Proliferation Assay

– More quantitative than counting cells on a hemocytometer (Less variable, no clumps)

– Fluorescent dye binds to nucleic acid in cell

Resources•  Mr. Mark Krotec, PTEI•  Mrs. Cassie Sirockman-Bell•  Dr. Phil Campbell•  Conrad M. Zapanta, Ph.D. Biomedical Engineering

Laboratory, Carnegie Mellon University •  Dr. Mark Seraly

www.PTEI.orghttp://www.ncbi.nlm.nih.gov/pubmed/19126049https://www.aad.org/dermatology-a-to-z/diseases-and-treatments/i---l/isotretinoinhttp://www.nlm.nih.gov/medlineplus/druginfo/meds/a681043.htmlwww.ipledgeprogram.comhttps://books.google.com/books?id=4y9ltnfmtlgC&pg=PA156&lpg=PA156&dq=dosage+amount+isotretinoin+chemo&source=bl&ots=pIJIg4VWpk&sig=NWGbLp9j4r4yA3axz_jqft5W-Dk&hl=en&sa=X&ved=0ahUKEwiOvMuD67nKAhWCSSYKHVrUCUcQ6AEIQTAG#v=onepage&q=dosage%20amount%20isotretinoin%20chemo&f=false

Day1ANOVA

Analysis of Variance (One-Way)

Summary Groups Sample size Sum Mean Variance

Control 8 1,493,000. 186,625.721,982,142

.85714

Low 8 415,000. 51,875.118,410,714

.28571

High 8 779,000. 97,375.170,839,285

.71429

ANOVA Source of Variation SS df MS F p-level F crit

BetweenGroups 7.51823E+10 2 3.75912E+10 111.520886.53022E-12 4.74039

WithinGroups7,078,625,000

. 21 337,077,380.95238

Total 8.2261E+10 23

Analysis of Variance (One-Way)

Summary Groups Sample size Sum Mean Variance

Control 8 1,888,000. 236,000.910,000,000

.

Low 8 1,592,000. 199,000.515,142,857

.14286

High 8 1,580,000. 197,500.415,428,571

.42857

ANOVA Source of Variation SS df MS F p-level F crit

BetweenGroups

7,609,333,333.33333 2

3,804,666,666.66667 6.20133 0.00765 4.74039

WithinGroups 1.2884E+10 21 613,523,809.52381

Total 2.04933E

+10 23

Day3ANOVA